Methods and compositions for obtaining mature dendritic cells

ABSTRACT

We describe an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The first step or “priming” phase is a culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4 to produce immature dendritic cells. The second step or “differentiation” phase requires the exposure to dendritic cell maturation factor such as monocyte conditioned medium. Using this two-step approach, substantial yields are obtained. The dendritic cells derive from this method have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. The mature dendritic cells produced according to this invention are useful for activating T cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 08/600,483 filedFeb. 12, 1996, the entire contents of which is incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with the United States Government support underNIH grants AR-39552, AR-42557 and AI-24775. The United States Governmenthas certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

This invention relates to methods and compositions useful for activatingcells of the immune system. The methods and compositions provided bythis invention are useful for causing the maturation ofnon-proliferating immature dendritic cells to mature dendritic cellscapable of processing and presenting antigen. This invention alsorelates to culture mediums which promote maturation of immaturedendritic cells to mature dendritic cells. In addition, this inventionrelates to assays useful for detecting the presence in a test substanceof a dendritic cell maturation factor.

BACKGROUND OF THE INVENTION

Dendritic cells are specialized antigen presenting cells, critical foreliciting T cell mediated immune responses(Steinman, 1991; Caux et al.1995a; Hart and McKenzie, 1990; Austyn, 1987). These specialized antigenpresenting cells elicit both CD4+ helper cells (Inaba et al. 1990a;Inaba et al. 1990b; Crowley et al. 1990) and CD8+ killer cells (Porgadorand Gilboa, 1995; Mayordomo et al. 1995; Zitvogel et al. 1995) in vivo.

Because of the potent activity of dendritic cells to activate T cells,the art, e.g. Flanand et al., Env. J. Immunol., 24:605–610, 1994, hasaccepted the characterization of dendritic cells as “nature's adjuvant”.It is therefore desirable to be able to use dendritic cells to processand present protein antigens as a means of modulating an individual'simmune response, and in particular to activate an individual's T cellsin connection with the treatment or prevention of disease.

The use of primed dendritic cells to activate cytotoxic T cells has beenreported. For example, Paglia et al., recently reported that murinedendritic cells cultured from bone marrow precursors and exposed toantigen in vitro provide effective resistance to challenge with livetumor cells. Paglia et al., “Murine Dendritic Cells Loaded In Vitro WithSoluble Protein Prime Cytotoxic T Lymphocytes Against Tumor Antigen InVivop”, J. Exp. Med., 183:317–322 (1996).

The in vitro observations and the murine results have recently beenextended to the treatment of humans with B cell lymphoma using maturedendritic cells which were primed in vitro with tumor antigens. Hsu etal., “Vaccination of Patients With B-cell Lymphoma Using AutologousAntigen-Pulsed Dendritic Cells”, Nature Med., 2:52–58 (1996). Accordingto this report, the antigen used to prime the dendritic cells wasidiotypic antibody obtained from hybridomas made from the fusion oflymph node tumor cells with a mouse cell line. All of the four patientsinvolved in this study exhibited significant antitumor idiotype PBMCproliferative responses. Positive clinical responses were also observedincluding one patient who experienced complete tumor regression. Hsu etal., however, express uncertainty whether dendritic cells obtained fromexpansion of dendritic cell cultures in the presence of GM-CSF, TNF-α orIL-4 would function equivalently in their ability to process and presentantigen or to stimulate cellular immune responses as the freshlyisolated cells.

Prior studies have identified proliferating dendritic cell progenitorswithin the small CD34+ subfraction of cells in human blood (Inaba et al.1992; Caux et al. 1994; Caux et al. 1992; Caux et al. 1995). Methodshave been developed for expanding these proliferating cells in cultureto obtain sufficient numbers of cells to be useful for priming withantigen and administering to an individual to activate an immuneresponse. Steinman et al. International patent application WO 93/20185.These dendritic cells can be stimulated with cytokines, particularlyGM-CSF and, optionally TNFα or other cytokines, to develop into potentdendritic cells over 1–2 weeks in culture (Caux et al. 1992; Inaba etal. (1992)). Although useful dendritic cells can be produced from theproliferating precursors, it is desirable to obtain alternative methodsof obtaining suitable numbers of mature dendritic cells for therapeuticpurposes where proliferating progentitors are infrequent.

The removal of monocytes and lymphocytes from human blood has uncovereda small population of nonproliferating progenitors that requirecytokines to develop into typical dendritic cells. These progenitorcells exist at a concentration of at most about 10⁶ cells per 450–500 mlof blood (O'Doherty et al. 1994; O'Doherty et al. 1993; Thomas et al.1993). More recently, the combination of GM-CSF and IL-4 has been shownto facilitate the generation of significantly larger numbers ofdendritic cells from adherent blood mononuclear fractions, about 3–8×10⁶per 40 ml of blood (Romani et al. 1994; Sallusto and Lanzavecchia, 1994both of which are incorporated herein by reference). However, we havenow determined that when the cytokines are removed, the cells revert toan adherent and less stimulatory state, that is, they do not have theproperties of mature, stable dendritic cells. If the latter reversionwere to take place in vivo during adoptive immunotherapy, the cellscould be ineffective as adjuvants. In addition, it is desirable todevelop a culture system independent of fetal calf serum.

Engleman et al. International Patent Application WO 95/34638 refers to amethod of activating an immune response in a human patient byadministering to the patient dendritic cells primed with antigen.Engleman et al. however refer to using dendritic cells isolated from theindividual which exist in small numbers. Accordingly, it would bedesirable to be able to obtain dendritic cells from a large populationof cells present in an easily accessible tissue such as blood.

Two antigens, have recently been reported which, in addition to otherantigens or phenotypic characteristics, distinguish mature dendriticcells from other types of white blood cells. Zhou and Tedder (Zhou andTedder, 1995) reported that CD83, a member of the immunoglobulinsuperfamily that was cloned from an EBV induced B cell library, isexpressed on dendritic cells in blood cells that were cultured for 2days. This culture period is sufficient to allow a small subset ofimmature dendritic cells to mature (O'Doherty et al. 1993). CD83 hasalso been detected on some presumptive dendritic cells in the T cellareas of lymphoid organs, and on some B cells in germinal centers (Zhouet al. 1992). Langhoff and coworkers found that p55, an actin bundlingprotein, also marks the dendritic cells that are found in 2 day culturesof human blood (Mosialos et al. 1995). p55 is an intracellular proteinthat was discovered as an EBV induced host cell product. It is expressedby interdigitating cells and at high levels in the brain (Mosialos etal. 1995).

SUMMARY OF THE INVENTION

This invention provides methods of preparing large numbers of stable,mature dendritic cells. The stable mature dendritic cells provided bythis invention retain characteristics of a mature phenotype, includingexpression of dendritic cell markers p55 and CD83 and high levels ofaccessory molecules like CD86 and CD40, even when removed from contactwith cytokines which promote the maturation of pluripotential peripheralblood mononuclear cells (PBMCs) to non-stable immature dendritic cells.As used herein, “stable” refers to retention of a mature dendritic cellphenotype for three days when mature dendritic cells are cultured in theabsence of cytokines. Dendritic cells are considered “mature” accordingto this invention when they have an increased expression of one or morephenotypic markers, such as for example non-adherence to plastic andCD86 or other antigen markers, associated with the accessory function ofdendritic cells following contact with a dendritic cell maturationfactor.

In one embodiment of the invention, mature dendritic cells are producedin vivo or in vitro from immature dendritic cells derived from PBMCpluripotential cells having the potential of expressing eithermacrophage or dendritic cell characteristics. The method, according tothe invention, comprises contacting the immature dendritic cells with adendritic cell maturation factor. The dendritic cell maturation factor,may actually be one or more different substances, and may be provided bysubstances including, but not limited to PBMC conditioned media,maturation factors purified from the conditioned medium, and SACS (fixedStaphylococcus aureus Cowan 1 strain (Pansorbin)). Culture of thepluripotential cells with cytokines such as for example, a combinationof GM-CSF and IL-4 or IL-13 stimulates the differentiation of thepluripotential cells to immature dendritic cells.

The method provided by this invention is particularly useful with humancells and provides sufficient numbers of mature dendritic cells to beuseful for priming with antigen in vitro and activating an individual'sT cells by administering the primed dendritic cells to the individual,or activation of T cells in vitro which are then administered to theindividual. Accordingly, another aspect of this invention is a method ofactivating an individual's T cells comprising:

-   -   a) obtaining a population of pluripotential cells having the        potential of expressing either macrophage or dendritic cell        characteristics from an individual and placing them in culture;    -   b) contacting the pluripotential cells of step a with at least        one cytokine to produce immature dendritic cells;    -   c) contacting the immature dendritic cells with a dendritic cell        maturation factor for a time sufficient to cause said immature        dendritic cells to stably express dendritic cell        characteristics;    -   d) contacting the dendritic cells obtained from step c with an        antigen to produce primed dendritic cells;    -   e) exposing T cells to the primed dendritic cells of step d to        activate said T cells. It is to be understood that one or more        of the above steps may be combined so as to occur concurrently,        such as for example, steps c and d.

Activation of T cell responses against a wide variety of antigens isfacilitated by this invention. Such antigens include, but are notlimited to viral, bacterial, tumor and self antigens such as antigenreceptors, e.g. T cell receptors. Accordingly, the dendritic cellsprepared according to this invention are useful for the prevention andtreatment of various diseases including infectious disease, cancer andautoimmune disease.

The methods of promoting maturation of dendritic cells to maturedendritic cells using cytokines and a dendritic cell maturation factor,and the subsequent activation of T cells, may also be applied toindividuals in vivo. According to this embodiment, an individual may beadministered cytokines, e.g. GM-CSF and IL-4, or G-CSF to stimulate invivo the production of immature dendritic cells. A dendritic cellmaturation factor, such as conditioned medium, or a substance whichstimulates the release of a dendritic cell maturation factor from PBMCs,may also be administered to an individual to stimulate in vivomaturation of immature dendritic cells, the population of which mayoptionally have been increased through prior administration ofcytokines.

This invention also encompasses the mature dendritic cells preparedaccording to the method of the invention and pharmaceutical compositionscomprising the mature dendritic cells and a physiological carrier. Suchcarriers may include the media described below.

Another aspect of this invention is a culture medium useful for causingthe maturation of immature dendritic cells to phenotypically stable,mature dendritic cells. The culture medium of the invention comprises amixture of salts, carbohydrates and vitamins at physiologicconcentrations typical of commercially available culture medium; as wellas about 1–5%, and more preferably 1% human serum or plasma; GM-CSF andone or more cytokines which promote maturation of the PBMC phenpotentialcells to immature dendritic cells and in amounts together sufficient topromote maturation of pluripotential PBMC to immature dendritic cells;and a sufficient concentration of a dendritic cell maturation factor tocause the maturation of immature dendritic cells to stable maturedendritic cells. This method can also be used with serum-free mediumsuch as for example, XVIVO-20 or AIM-V.

Another aspect of this invention is an assay to detect a dendritic cellmaturation factor. Such an assay is useful for the identification andproduction of the factor present in PBMC conditioned medium which causesthe maturation of immature dendritic cells to the mature dendritic cellsprovided by this invention. This assay comprises contacting a testsample with a culture of unstable, immature dendritic cells derived froma population of pluripotential cells having the potential of expressingeither macrophage or dendritic cell characteristics and which expresscharacteristics of immature dendritic cells when cultured in a mediumcontaining at least one cytokine. The presence of a dendritic cellmaturation factor in the test substance is then determined by detectingthe maturation of the immature dendritic cells in response to the testsubstance.

It is an object of this invention to provide methods and compositionsuseful for activating an individual's T cells against specific antigens.The activation of an individual's T cell is useful for the prevention ortreatment of disease, for example killer cells to treat or vaccinateagainst cancer or infection.

It is another of this invention to provide methods useful for causingthe stable maturation of immature dendritic cells to mature dendriticcells suitable for administering to an individual for the purpose ofactivating their immune response, and in particular, T cells.

Another object of this invention is to provide mature dendritic cellscultured in vitro from non-proliferating pluripotential cells.

Another object of this invention is to provide culture mediums whichpromote the maturation of immature dendritic cells to stable maturedendritic cells.

Yet another object of this invention is to provide an assay useful fordetecting the presence in a test sample of a dendritic cell maturationfactor.

Another object of this invention to provide methods for stimulating thematuration of dendritic cells in vivo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Conditioned medium (CM) is required to ensure the maturation ofdendritic cells from progenitor cells. ER− cells were cultured for 7days in RPMI medium containing 1% plasma in the presence of GM-CSF andIL-4. Cells were then transferred to fresh plates and cultured for 4days in the presence [open squares] or absence [closed circles] of CM.Control cultures were ER− cells cultured in Teflon beakers for 11 days[open circles]. T cell stimulatory function [primary allogeneic MLR] ofthe various APC populations is shown.

FIG. 2: Cytofluorographic analysis of dendritic cells grown in thepresence of GM-CSF/IL-4 and CM. Dead cells and contaminating lymphocyteswere excluded by light scatter properties. Dot plots of the remainingcells are shown. Isotype controls are shown in the left panel.

FIG. 3: Morphology and phenotype of blood derived dendritic cells andmacrophages. Dendritic cells [left panel] were generated from normalblood ER− cells in the presence of GM-CSF/IL-4 and CM, cytospun ontoglass slides and stained with a panel of mAbs [see Methods]. Macrophageswere syngeneic ER− cells, enriched by plastic adherence and cultured fortwo weeks in Teflon beakers. CD8 serves as the isotype control antibody.Dendritic cells are distinguishable from macrophages by their dendriticshapes, lack of CD14 expression, high p55 expression and perinuclearpattern of CD68 expression. Black arrows in the right panel point topresumptive dendritic cells contaminating the macrophage populations.

FIG. 4: Attempts to replace conditioned medium with cytokines. ER− cellswere cultured in GM-CSF/IL-4 for 7 days and then transferred to freshtissue culture plates. The cells were supplemented with 50% CM [blackcircles] in the absence or presence of polyclonal neutralizingantibodies to TNFα, IL-1β and IL-6 [1–5 μg/ml]. Cocktail refers to thecombination of the various antibodies. Some cultures received TNF alpha[20 ng/ml] or IL-1 beta [10 ng/ml] instead of CM. After 4 days [day 11of culture], the cells were evaluated for T cell stimulatory activity inan allo MLR. Results are shown as ³H-TdR incorporation [cpm×10³¹ ³] andare averages of triplicates.

FIG. 5: Dendritic cell progenitors are enriched in the CD14 hipopulation. ER− cells from two different sources were sorted into CD14hi and lo populations as described in Methods. Unsorted and varioussorted fractions were analyzed for T cell stimulatory activity in anMLR. In [A], the CD14 hi cells were irradiated with 3,000 rads, 137 Cssource. In [B], the CD14 hi cells were irradiated with 1,500 rads.Results are shown as ³HTdR incorporation [cpm×10⁻³] and are averages oftriplicates.

FIG. 6: Morphological stability of mature dendritic cells.Lymphocyte-depleted PBMC were cultured for 10 days in FCS-containingRPMI medium in the presence of GM-CSF and IL-4. Phase contrastmicrographs were taken to illustrate the morphology of the cells. [A] NoCM was added during the 10 day culture. Cells were washed on day 10 andrecultured without cytokines for 3 additional days. They re-adherefirmly and have the appearance of macrophages. [B] CM was added from day7–10. The cells acquire many lamellipodia or veils and are nonadherent.[C,D] CM was added from day 7–10, the cells were washed and returned toculture for 3 days without CM or cytokines. The morphology of maturedendritic cells persists both in RPMI/10% FCS [C] and X-VIVO20/serum-free [D] media. A, x275; B–D, x550.

FIG. 7: Comparative cytoflurographic analysis of mature vs. immaturedendritic cells. Lymphocyte-depleted PBMC were cultured for 7 days withGM-CSF and IL-4 followed by a maturation period from day 8 to day 10 inthe presence of CM. The resulting fully mature dendritic cells werecompared to corresponding immature dendritic cells that had not beentreated with CM. In all cases mature dendritic cells express CD83 andenhanced levels of CD86, and they lose CD115 expression. [A,A′]Dendritic cells cultured from day 7 to day 10 without [A] or with [A′]CM in RPMI/FCS are shown. Fluorescence on the x-axis represents HLA-DRexpression; y-axis shows the indicated antibody staining. Dot plots arefrom cells gated on the basis of their light scatter properties[FSC/SSC]. [B,B′] Cells were analyzed on day 7[B] and on day 10, i.e.,after maturation in the presence of CM [B′]. Culture medium was RPMI/1%human plasma; lymphocytes were depleted with immunomagnetic beads.Histograms are from cells gated on the basis of their light scatterproperties [FCS/SSC]. [C,C′-left] Like B,B′, but lymphocytes wereinitially depleted by E-rosetting. [C,C′-middle] like B,B′, but AIM-Vmedium/1% human plasma was used. [C,C′-right′ like A,A′, co-2b,isotype-matched control IgG2b [for CD83, 86, 14]; co-rat, control forrat IgG1 [for CD115]; FSC/SSC, forward scatter/side scatter. All markersare set according to staining with isotype-matched control antibodies.

FIG. 8: Fixed staphylococci [SACS] are required to ensure the maturationof dendritic cells from progenitor cells. Lymphocyte-depleted PBMC werecultured for 7 days in RPMI medium supplemented with FCS andGM-CSF/IL-4. Cells were then transferred to fresh plates and culturedfor 3 more days in the presence [closed squares] or absence [opensquares] of SACS. T cell stimulatory function in the primary allogeneicMLR of the dendritic cells matured in SACS containing medium is markedlyenhanced. Neither IL-1β [in A] nor IL-15 [in B] that were given insteadof SACS could fully reconstitute the effect.

FIG. 9: Morphology and motility of mature dendritic cells grown in thepresence of GM-CSF/IL-4 and CM. Dendritic cell progenitors were primedfor 7 days in the presence of GM-CSF and IL-4 followed by a 3 daymaturation period in the presence of CM. Culture medium was RPMIsupplemented with 1% autologous human plasma. Note the numerous thincytoplasmic processes (“veils”). Photographs of the same field taken atintervals of 15 seconds show that the veils move. Magnification: ×550.

FIG. 10: Phagocytosis of latex beads. Dendritic cells were grown in thepresence of GM-CSF and IL-4 until day 8. GCS-containing RPMI medium wasused. Cells in the upper panel were exposed to latex beads for 24 hoursin the absence of CM. Latex is taken up into the cells (arrows). In thelower panel cells were first allowed to mature in the presence of CMfrom day 7 to day 10 and were then exposed to latex. Little or no latexis phagocytosed into mature, “veiled” cells. Only a contaminatingadherent cell has accumulated beads. ×300.

FIG. 11: Immunostimulatory functions of mature dendritic cells.Dendritic cells were cultured in the presence of GM-CSF and IL-4 untilday 7 and further on until day 10 with [closed symbols] or without [opensymbols] CM. RPMI was supplemented with FCS in A and D–F, and with 1%autologous human plasma in B and C. [A] Note pronounced enhancement of Tcell stimulatory capacity in the MLR following exposure to CM. Twoindependent experiments are shown. [B] Fully mature dendritic cellsgrown in RPMI supplemented with 10% FCS or 1% autologous human plasmawere compared to each other. [C] Fully mature dendritic cellsefficiently stimulate naive cord blood T cells. [D] Tetanus toxoidprotein [1 μg/ml] is presented to a peptide-specific T cell clone.CM-induced maturation leads to a decrease in antigen processingcapacity. Triangles in D indicate the [lack of] response of clone cellsto APC in the absence of antigen. Values are means of triplicate wellsand are expressed as cpm×10⁻³. [E,F] Peptide-pulsed mature dendriticcells elicit T cell lines that lyse autologous target cells in a peptidedose-dependent [E] and MHC-restricted [F] manner. anti-cl. II, anti-cl.I, anti-MHC class II and I mAb's W6/32 and L243, respectively.

FIG. 12: Dendritic cell progenitors reside in a CD34-negativepopulation. PBMC of a cancer patient who had been treated with G-CSFwere passed through an anti-CD34 immunoaffinity column. The originalunseparated population [solid lines] and the CD34-depleted population[dashed lines] were cultured in RPMI\FCS with GM-CSF and IL-4 until d7and further on from d7 to d10 in the presence [closed symbols] orabsence [open symbols] of CM. Both populations gave rise to dendriticcells that matured upon exposure to CM.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to methods and compositions useful for promotingthe maturation of immature dendritic cells to a mature and stablephenotype as well as to the stable mature dendritic cells. The maturedendritic cells are useful as antigen presenting cells (APCs) whichactivate other immune cells including antigen specific helper and killerT cells. Thus, this invention also provides methods and compositionsuseful for the treatment of disease.

The method of producing mature dendritic cells according to thisinvention comprises contacting immature dendritic cells with a dendriticcell maturation factor. We have found such a dendritic cell maturationfactor in conditioned medium obtained from cultures of peripheral bloodmononuclear cells (PBMCs). Immature dendritic cells may be derived fromPBMCs by culturing PBMCs with cytokines, such as, for example, acombination of GM-CSF and IL-4, which promote their differentiation toimmature dendritic cells. Surprisingly, we have determined that unlessthey are exposed to a dendritic cell maturation factor, removal of thecytokines from contact with the cells causes the cells to revert back toa pluripotential cell having characteristics similar to macrophages.

The response of immature dendritic cells to contact with the maturationfactor is an increased expression of CD83 and p55 dendritic cellmarkers; strong expression of antigen presenting MHC class I and IIproducts; and expression of several accessory (adhesion andco-stimulatory) molecules including CD40, CD54, CD58, CD80 and CD86. Adecrease in expression of CD115 as well as CD14, CD68 and CD32, whichare markers associated with immature dendritic cells, is also observedas a result of carrying out the method of this invention. In addition,this phenotype remains stable for up to three days even after removal ofcytokines used to promote cell maturation.

Sources of Pluripotential Cells

The pluripotential cells, from which the immature dendritic cells foruse in this invention are derived, are present in blood as PBMCs.Although most easily obtainable from blood, the pluripotential cells mayalso be obtained from any tissue in which they reside, including bonemarrow and spleen tissue. These pluripotential cells typically expressCD14, CD32, CD68 and CD115 monocyte markers with little or no expressionof CD83, p55 or accessory molecules such as CD40 and CD86. When culturedin the presence of cytokines such as a combination of GM-CSF and IL-4 orIL-13 as described below, the pluripotential cells give rise to theimmature dendritic cells.

Methods of obtaining PBMCs from blood, such as differentialsedimentation through an appropriate medium, e.g. Ficoll-Hypaque[Pharmacia Biotech, Uppsala, Sweden], are well known and suitable foruse in this invention. In a preferred embodiment of the invention, thepluripotential cells are obtained by depleting populations of PBMCs ofplatelets, and T and B lymphocytes. Various methods may be used toaccomplish the depletion of the non-pluripotential cells. According toone method, immunomagnetic beads labelled with antibodies specific for Tor B lymphocytes, either directly or indirectly may be used to removethe T and B cells from the PBMC population. T cells may also be depletedfrom the PBMC population by rosetting with neuraminidase treated redblood cells as described by O'Dherty (1993), which is incorporatedherein by reference.

To produce 3 million mature dendritic cells, it is necessary to processabout 40 mls of blood. 4 to 8×10⁷ pluripotential PBMC give rise toapproximately 3 million mature dendritic cells.

Culture of Pluripotenial PBMCs to Produce Immature Dendritic Cells

The immature dendritic cells for use in this invention are post-mitoticbut not yet terminally differentiated to either a macrophage ordendritic cell phenotype. Cultures of immature dendritic cells may beobtained by culturing the pluripotential cells in the presence ofcytokines which promote their differentiation. A combination of GM-CSFand IL-4 at a concentration of each at between about 200 to about 2000U/ml, more preferably between about 500 and 1000 U/ml, and mostpreferably about 800 U/ml (GM-CSF) and 1000 U/ml (IL-4) producessignificant quantities of the immature dendritic cells. A combination ofGM-CSF (10 ng/ml) and IL-4 (10–20 ng/ml) has been found to be usefulwith this invention. It may also be desirable to vary the concentrationof cytokines at different stages of the culture such that freshlycultured cells are cultured in the presence of higher concentrations ofIL-4 (1000 U/ml) than established cultures (500 U/ml IL-4 after 2 daysin culture). Other cytokines such as IL-13 may be found to substitutefor IL-4.

Methods for obtaining these immature dendritic cells from adherent bloodmononuclear fractions are described in Romani et al. (1994); andSallusto and Lanzavecchia, 1994) both of which are incorporated hereinby reference. Briefly, lymphocyte depleted PBMCs are plated in tissueculture plates at a density of about 1 million cells/cm² in completeculture medium containing cytokines such as GM-CSF and IL-4 atconcentrations of each at between about 800 to 1000 U/ml and IL-4 ispresent at about 1000 U/ml.

Various mediums are suitable to initially culture PBMCS. Typical mediafor use in this invention comprise physiologic inorganic salts,carbohydrates including sugars, amino acid, vitamins and othercomponents known to those in the art. Preferred media formulationsinclude, for example, RPMI 1640, X-VIVO 20, AIM-V, Hybricare andIscove's. More preferably the medium is RPMI 1640 or X-VIVO 20 or AIM-V.Most preferably, the medium is RPMI 1640.

It is desirable to avoid the use of fetal calf serum (FCS) in culturesfor human use. The presence of bovine proteins may cause unwantedsensitization of the recipient against bovine proteins and it istherefore desirable to substitute FCS with human serum or plasma in anamount sufficient to maintain cell viability. The use of autologous,non-heat-inactivated human plasma at a concentration of about 1% ispreferred. Using RMPI 1640 supplemented with 1% autologous human plasma,a final yield of between about 0.8 to 3.3×10⁶ CD83+ mature dendriticcells is obtainable from 40 ml of blood.

Another source of immature dendritic cells are cultures of proliferatingdendritic cell precursors prepared according to the method described inSteinman et al. International application PCT/US93/03141, which isincorporated herein by reference. Since the dendritic cells preparedfrom the CD34+ proliferating precursors mature to dendritic cellsexpressing mature characteristics it is likely that they also passthrough a development stage where they are pluripotential. By treatingthese cultures with a dendritic cell maturation factor as describedbelow, the efficiency of obtaining mature dendritic cells fromproliferating precursors may also be improved.

Maturation of Immature Dendritic Cells

The mature dendritic cells are prepared according to this invention bycontacting the immature dendritic cells with a dendritic cell maturationfactor. As referred to herein, the dendritic cell maturation factor mayactually be one or more specific substances which act in concert tocause the maturation of the immature dendritic cells. Such a factor hasbeen determined to be present in PBMC conditioned medium, preferrablymonocyte conditioned medium. Another means of obtaining dendritic cellmaturation factors are to treat PMBCs with substances which stimulatethe release of such factors. Substances such as immunoglobulin or SACS(also referred to as Pansorbin) are useful for causing monocytes torelease cytokines.

After the cells have been cultured for a sufficient time to express somecharacteristics of dendritic cells, preferably about 6 to 10 days, PBMCconditioned media is added to the culture medium at a finalconcentration of between about 10 to 50%. More preferably between about25 and 50%. Maturation of the immature dendritic cells to the maturephenotype may occur without the presence in the medium of the cytokinesused to stimulate maturation of the pluripotential cells to immaturedendritic cells. Typically, the immature dendritic cells are obtainedafter about 6 to 7 days in culture with the cytokines. Preferably theimmature dendritic cells are subcultured prior to addition of thedendritic cell maturation factor, e.g., conditioned medium.

In a preferred embodiment, the immature dendritic cells which arenon-adherent cells are harvested and subcultured in the presence of thedendritic cell maturation factor present in the conditioned medium, andoptionally with cytokines such as GM-CSF and IL-4. The mature dendriticcells are typically obtained within approximately three days followingaddition of the conditioned medium.

Variations in the culture protocol which are also considered within thescope of this invention may include, but not be limited to, changes incytokines used to promote differentiation of PBMCs to immature dendriticcells, concentrations of cytokines and timing of the addition of thedendritic cell maturation factor present in the conditioned medium.

Characterization of Conditioned Medium

The conditioned medium for use in this invention may be made byculturing PBMCS, preferrably monocytes in basic growth medium.Preferably the cells for producing conditioned medium are cultured inthe presence of a stimulus which stimulates the release of factors fromthe donor cells. An example of such a preferred factor is gamma globulinwhen bound to the culture substrate. Methods for producing PBMC(monocyte) conditioned medium have previously been described inO'Doherty et al. (1993 and 1994) which are incorporated herein byreference. PBMC or preferably T cell depleted, or more preferrably T andB cell depleted are cultured (about 5×10⁷ cells/100 mm plate) for about24 hours in medium (6–8 mls) containing 1% of plasma, preferrablyautologous plasmsa. Where conditioned medium is to be used forstimulating maturation of dendritic cells in vivo, the growth mediumshould be selected to be compatible with use in humans, e.g. X-VIVO orAIM-V and preferrably is used without serum or plasma. For use in humansthe conditioned medium is concentrated using standard procedures andadjusted to be isotonic so as to be suitable for administration in vivo.Using this method, equivalent concentrations of dendritic cellmaturation factor can be achieved in vivo, as in vitro.

In addition to gamma globulin as a stimulant of cytokine release fromthe culture PBMCs, other cytokine stimulators such as SACS (fixedStaphylococcus Aureus Cowan 1 strain, 2,01 mg/ml Ig-binding capacity:Pansorbin cells, Cat. No. 507861) may also be used. Because SACS isbacterially derived its use is less desirable than Ig.

In addition to stimulating the production of cytokines for producingconditioned medium, SACS may also be substituted for conditioned mediumas a dendritic cell maturation factor. When used to promote maturationof the immature dendritic cells, SACS is added to cultures at aconcentration sufficient to cause their maturation, preferably at about1:10000 dilution.

This invention provides evidence for a new pathway for dendritic celldevelopment. Prior studies have indicated that dendritic cells ariseeither from CD34+ proliferating progenitors (Inaba et al. 1992; Caux etal. 1992), or from nonproliferating “null” cells that lack monocyte andlymphocyte markers [CD3, CD14, CD16, CD19](O'Doherty et al. 1993). Bothof these progenitor populations are infrequent in human bloodrepresenting <0.1% and <1% of blood mononuclear cells, respectively.Sallusto and Lanzavecchia [personal communication] reason that monocytescan give rise to dendritic cells in the presence of FCS, but we findthat these cells are not fully mature in several respects. In contrast,large numbers of mature dendritic cells can develop, and in human plasmaor serum, when CM is added to the GM-CSF and IL-4 priming step. Theprecursors are radioresistant and primarily in the standard “monocyte”fraction [CD14 positive and plastic adherent]. It is possible that mostmonocytes are bipotential and can develop into either macrophages ordendritic cells depending upon the cytokines that are applied.Alternatively, only some of the “monocyte” fraction may have thepotential to develop into dendritic cells. At this time, our data areconsistent with the latter possibility since the total yield ofdendritic cells from radioresistant precursors represents about 5.5–16%of the starting number of monocytes.

Physiologic counterparts for the maturation over several days ofdendritic cells from nonproliferating blood progenitors can be proposedin at least three settings. First sizable numbers of dendritic cellstraffic in afferent lymphatics, and these cells derive from precursorsthat have been proliferating 3–7 days prior to detection in lymph (Masonet al. 1981; Pugh et al. 1983; Fossum, 1989a; Fossum, 1989b). Theseafferent lymph dendritic cells may derive from blood progenitor cellsthat undergo a priming phase. Second, dendritic cells are rapidlyinduced to migrate into tissues [lung, gut] in response topro-inflammatory stimuli e.g. LPS or infection (Watson et al. 1990;McWilliam et al. 1994). There they may undergo further maturation inresponse to cytokines that are produced locally by tissue macrophages.

Third, significant numbers of mature dendritic cells accumulate inrheumatoid synovial exudates during acute flares of arthritis (Zvaifleret al. 1985; Bhardwaj et al. 1988; Helfgott et al. 1988; Thomas et al.1994). These effusions are rich in GM-CSF, IL-4, IL-6 and TNF alpha,amongst other cytokines [reviewed in (Klareskog et al. 1995)]. Inaddition, the presence of substantial numbers of macrophages and immunecomplexes may provide a natural “conditioned medium” for the maturationand differentiation of dendritic cells from blood progenitors that enterinflamed joints.

Based on the data reported in Examples 1 and 2, dendritic cells culturedfrom peripheral blood precursors in GM-CSF and IL-4 for 6–7 days can beconsidered as immature dendritic cells. Although they have alreadyacquired relatively high levels of MHC and adhesion/costimulatormolecules as well as the capacity to stimulate resting T cells theystill lack markers for terminal dendritic cell differentiation such asCD83. They are however well equipped with the necessary prerequisitesfor the processing of native protein antigens. They can phagocytoseparticulate matter and they can efficiently process native tetanustoxoid protein into immunogenic MHC-peptide complexes recognized by atetanus peptide-specific T clone. In addition, Sallusto et al. (1995)described active uptake of soluble macromolecules by macropinocytosis,and we find uptake of latex particles as well. Immature dendritic cellsmay therefore be useful in immunization protocols that use nativeproteins or particles as antigens. One must keep in mind, however, thatthe immature dendritic cells generated with GM-CSF and IL-4, but withoutCM, are not stable. Upon withdrawal of cytokines they re-adhere andappear to revert back to monocytes. Such cells would therefore not be asuseful for therapeutic approaches. It will be preferred to inducematuration of antigen-pulsed dendritic cells by the method describedhere, i.e., exposure to monocyte-conditioned media.

Dendritic cells grown in GM-CSF and IL-4 for 7 days and exposed tomacrophage-conditioned media for another three days develop into maturedendritic cells. These cells have down-regulated their antigen uptakemechanisms and their processing capacity. CM-treated dendritic cellscorrespond morphologically, phenotypically and functionally to welldefined populations of mature dendritic cells, e.g., cultured epidermalLangerhans cells (Romani et al. 1989b); Tenunissen et al. 1990),cutaneous dendritic cells obtained by emigration from skin explants(Pope et al. 1995; Lenz et al. 1993), or blood dendritic cells obtainedby classical methods involving 36 hours of culture (Young and Steinman,1988). They are specialized in the sensitization of naive, resting Tcells. We show here that they can efficiently induce proliferation inallogeneic umbilical cord T cells which may be considered virgin Tcells. We also demonstrate that these mature dendritic cells can beloaded with an antigenic peptide and induce antigen-specific cytotoxic Tlymphocytes from populations of autologous PBMC or CD8+ T cells. Inaddition, mature dendritic cells in small numbers are able to elicitrapid and virus-specific CTL responses from quiescent autologous Tcells. Mature dendritic cells will be helpful in immunization protocolsthat employ antigenic peptides preferably to proteins. This appliesespecially to the field of tumor immunotherapy. Tumor-specific peptidesare being discovered at a fast rate (Boon et al. 1994).

Based on our data, the optimal method to generate mature dendritic cellsfor clinical purposes would be to deplete PBMC of lymphocytes withimmunomagnetic beads, culture them for 6 to 7 days in RPMI mediumsupplemented with 1% autologous human plasma and GM-CSF/IL-4, and inducethem to mature within another three days of culture by the addition ofconditioned medium that is produced by PBMC adhering to Ig-coated Petridishes. All these reagents are already approved [Anti-mouse Ig Dynabeadsand mouse anti-T and B cell mAb's /Baxter; GM-CSF/Sandoz;IL-4/Schering-Plough; lg/Biochemie-Sandoz] or have been used in clinicalstudies [RPMI medium, Lymphoprep].

Our method to generate dendritic cells from progenitor cells inautologous plasma will be useful in several respects. First, the methodis simple, reproducible and generates 1–3×10⁶ dendritic cells fromrelatively small, 40–50 ml blood samples. Second, critical features ofdendritic cells such as pathways of antigen presentation and T cellsignalling can be studied in the absence of foreign FCS derivedproteins. Third, one has ready access to cells that can be used asadjuvants to enhance protective immune responses in vivo. We find thatdendritic cells generated by the method described here and pulsed witheither influenza virus [live or heat inactivated] or immunodominantpeptides [Table 6] induce virus-specific CD8+ killer cell responses invitro.

Pulsing Dendritic Cells with Antigen

The mature dendritic cells prepared according to this invention areuseful for activating T cells against specific antigens. Severalantigens and methods for priming dendritic cells have been described andmay be adapted for use in this invention. See, for example Engleman etal. International patent application PCT/US95/07461; Hsu et al., NatureMed. 2:52–58 (1996); and Steinman et al. International applicationPCT/US93/03141 which are all incorporated herein by reference. Antigensassociated with fungal, bacterial, viral, tumor, or autoimmune (i.e.,self antigens) diseases are useful for priming dendritic cells toactivate T cells which aid in treating or preventing disease.

Where it is desirable for cells to take up antigen by phagocytosis, itis preferable to add antigen to the cultures of immature dendritic cellsprior to addition of the dendritic cell maturation factor. Phagocytosismay be desirable when particulate antigens, or immune complexes areused. In most cases it is sufficient to expose antigen to the dendriticcells after they have attained the mature phenotype or while they areexposed to the dendritic cell maturation factor to attain the maturephenotype. This method is preferred when soluble peptide antigens areused.

For the purpose of priming cells typically approximately 1 to 5×10⁶cells are exposed to antigen at a concentration of between about 10 μMto about 10 μM, inclusive. More preferably about 1 μM antigen to about 3million cells is used. The dendritic cells are cultured in the presenceof the antigen for a sufficient time to allow for uptake andpresentation. Typically uptake and processing can occur within 24 hoursbut longer (up to and including 4 days) or shorter (about 1–2 hours)periods may also be used.

For activating T cells in an individual between about 2×10⁵ and 2×10⁹more preferably between about 1 million and 10 million mature dendriticcells should be administered to the individual. The dendritic cellsshould be administered in a physiologically compatible carrier which isnontoxic to the cells and the individual. Such a carrier may be the cellgrowth medium described above. The mature dendritic cells preparedaccording to this invention are particularly potent at activating Tcells. For example, using prior methods of obtaining dendritic cells theratio of dendritic cells to T cells necessary for strong T cellactivation is about 1 dendritic cell to 30 T cells whereas the ratioaccording to this invention is about 1 to 1000. Thus, fewer dendriticcells are required. For activating T cells in vitro the ratio ofdendritic cells to T cells is between about 1:10 and 1:1000. Morepreferably between 1:30 and 1:150. Between approximately 10⁸ and 10⁹activated T cells are administered back to the individual to produce aresponse against an antigen.

Dendritic cells may be administered to an individual using standardmethods including intravenous, intraperitoneal, subcutaneously,intradermally or intramuscularly. The homing ability of the dendriticcells facilitates their ability to find T cells and cause theiractivation.

The methods of this invention are particularly well suited for useagainst tumors from which tumor specific antigens are obtainable orwhich express a mutated protein. Preferrably, the antigen is a moleculerequired by the tumor cells to be malignant, for example altered ras.Non-limiting examples of tumors for which tumor specific antigens havebeen identified include melanoma, B cell lymphoma, uterine or cervicalcancer. An example of a melanoma antigen which could be considered foruse to prime dendritic cells is the human melanoma specific antigen gp75antigen (Vijayasardhi S. et al., J. Exp. Med., 171:1375–1380 (1990). Anexample of an antigen useful for targeting cells against cervical canceris papilloma virus. Tumor specific idiotypic protein derived from B celllymphomas has been reported to prime dendritic cells which activate Tcells against the tumor cells. Hsu et al. (1966), supra. In addition totumor antigens, viruses may also be used to prime the dendritic cells.Viruses may also be used to cause dendritic cells to present viralantigens, or they may be engineered to express non-viral proteins whichare then processed and presented by the dendritic cells. Non-limitingexamples of viruses which may be used to prime dendritic cells include,but are not limited to, influenza, HIV, CMV, EBV, human papilloma virus,adenovirus, HBV, HCV and vaccinia. Isolated viral proteins, inactivatedor attenuated virus may also be used. Non-limiting examples of bacterialor protozoan antigens include tetanus toxoid, BCG, malaria antigens andleishmania antigens. Specific antigens associated with autoimmunedisease such as immune receptors may also be used to prime dendriticcells to be used to activate T cells against such antigens.

Method of Assaying for a Dendritic Cell Maturation Factor

This invention provides an assay useful for identifying the dendriticcell maturation factor present in a test substance such as monocyteconditioned medium. This assay is based on the ability of immaturedendritic cells to mature and express a stable phenotype after havingbeen contacted with a dendritic cell maturation factor. Thus, the assaycomprises contacting a culture of immature dendritic cells with a testsubstance and detecting the maturation of the immature dendritic cellsin response to the presence of the test substance. The immaturedendritic cells for use in this assay are derived from a population ofpluripotential cells, such as human PBMCs having the potential ofexpressing either macrophage or dendritic cell characteristics and whichexpress characteristics of immature dendritic cells when cultured in amedium containing cytokines (e.g., GM-CSF and IL-4). Maturationassociated with the presence of a dendritic cell maturation factor maybe confirmed, for example, by detecting either one or more of 1) anincrease expression of one or more of p55, CD83, CD40 or CD86 antigens;2) loss of CD115, CD14, CD32 or CD68 antigen; or 3) reversion to amacrophage phenotype characterized by increased adhesion and loss ofveils following the removal of cytokines which promote maturation ofPBMCs to the immature dendritic cells. The assay described above may beused in conjunction with biochemical techniques to identify thedendritic maturation factor present in monocyte CM. In addition, itprovides a useful bioassay to confirm the activity of such a factor onceit is identified or purified in connection with its manufacture.

EXAMPLES Example 1

Methods

Culture Medium

RPMI 1640 supplemented with 20 μg/ml of gentamicin, 10 mM HEPES, andeither 10% heat-inactivated FCS [Gemini Bioproducts, Calabasas, Calif.]or 1% and 10% autologous plasma [heparinized].

Cytokines

We were generously supplied with rhGM-CSF [1×10⁵ U/μg] by Kirin BreweryCo., Maebashi, Gunma, Japan, and rhIL-4 [0.5×10⁵ U/μg] by Immunex Corp.rhIL-12 [2×10⁷ U/mg] was provided by the Genetics Institute, Boston,Mass. The recombinant human cytokines IL-15, IL-1, IL-6, TNF alpha werepurchased from R and D Corp [Minneapolis, Minn.].

Generation of Dendritic Cells from Human Blood

Peripheral blood was obtained from normal donors in heparinizedsyringes, and PBMC isolated by sedimentation in Ficoll-Hypaque[Pharmacia Biotech, Uppsala, Sweden]. T cell-enriched [ER+] and Tcell-depleted [ER−] populations were prepared by rosetting withneuraminidase treated-sheep red blood cells as described (Carr and Kim,1993). Different starting populations of PBMC were plated in 3 mlvolumes in 6 well tissue culture dishes [Falcon] in complete medium.They consisted of [a] plastic adherent and nonadherent PBMC [obtainedafter 60 min adherence of 6×10⁶ PBMC/well]; [b] ER− cells; [c] ER− cellssorted on a FACStar^(Plus) into CD14 high or low cells. Groups [b]–[c]were plated at 2×10⁶/well. GM-CSF and IL-4 were added at finalconcentrations of 1000 U/ml on the initial day of culture. Cytokineswere replenished every other day [days 2, 4 and 6] by removing 0.3 ml ofthe medium and adding back 0.5 ml fresh medium with cytokines. On day 7,non-adherent cells were collected by moderately vigorous aspiration andanalyzed immediately, maintained in culture or transferred to new 6 wellplates. When cultured beyond 7 days, the cultures were supplemented withPansorbin, [1:10,000 dilution of 2 mg IgG/ml, Calbiochem, La Jolla,Calif.], or conditioned medium [final concentration 33 or 50% v/v, seebelow] at day 7.

Conditioned Medium

Ig coated bacteriologic plates [100 mm, Falcon, Lincoln Park, N.J.] wereprepared immediately prior to use by the addition of 4 ml of humangamma-globulin [10 mg/ml, Cappel Labs, Organon Teknika, Westchester,Pa.] for 1 min. The residual gamma-globulin was removed and saved forreuse at least 4–6 times. The plates were washed three times withphosphate buffered saline [PD] prior to use. T cell-depleted ER− cells[5×10⁷] were layered onto the Ig-coated bacteriologic plates for 1 hourin volumes of 6–8 ml. Non-adherent cells were washed off with gentleaspiration. The gamma-globulin adherent cells were incubated in freshcomplete medium with 1% autologous plasma at 37° C. for 24 h. The mediumwas collected and frozen at −20° C. prior to use. In some cases,conditioned medium was prepared from cells stimulated with pansorbininstead of human Ig.

Monoclonal Antibodies [mAbs]

MAbs towards the following antigens were used: HLA-DR, CD14, CD3,CD4/CD8, CD5, CD19, CD16, CD56, CD1a, CD54, CD58, CD25, CD45RO, CD11a,CD11b and CD11c, [Becton-Dickinson, Mountainview, Calif.], CD80 and CD86[IgG1, FITC conjugate, Pharmingen], CD83 [IgG1, PE conjugate, CoulterCorp, Miami, Fla.], CD68 [Dako, Carpinteria, Calif.], CD40, CD95/fas[Immunotech, Marseilles, France], p55 [K-2 clone, a gift from Dr. E.Langhoff; Mosialos et al. (1996)], mannose receptor [mAb 3.2PB1, a giftfrom Dr. A. Lanzavecchia], Lag anti-Birbeck granule associated antigen[a gift from Dr. S. Imamura], Ki 67 [MIB-1,IgG1, Dako]. Secondaryantibody was PE-conjugated F[ab′]2 goat anti-mouse IgG [gamma and lightchain, Tago, Burlingame, Calif.].

Polyclonal Neutralizing Antibodies

Goat anti-human polyclonal antibodies were purchased from R and D Corp,Minneapolis, Minn. They included antibodies toward TNFa, IL-1 beta, IL-1alpha, IL-12 and IL-6. Control antibody was goat IgG [R and D Corp.].

Cytofluorography and Cell Sorting

Cell populations were phenotyped with the panel of Mabs listed above andanalyzed on a FACScan. To obtain CD14+ cells, 2×10⁷ ER− cells werestained with PE-CD14 [Becton-Dickinson, Mountainview, Calif.] for 30 minon ice, washed extensively and then sorted on a FACStar^(Plus). BothCD14 high and CD14 low cells were evaluated for the ability to developinto dendritic cells.

Immunohistochemistry

Cytospins of various cell populations were prepared using a Cytospin 2centrifuge [Shandon, Inc., Pittsburgh, Pa.]. Slides were fixed inabsolute acetone for 5 min at room temperature and then incubated withmAbs for 45 min. Cytospins were then washed and incubated with 1:200dilution of biotinylated goat anti-mouse IgG [Boehringer MannheimBiochemicals, Indianapolis, Ind.] for 45 min, followed by a horseradishperoxidase [HRP]-biotin-avidin complex [ABC kit; Vector laboratories,Inc., Burlingame, Calif.] for 30 min. Non-bound HRP was then washed off,and the HRP reaction product was developed with H₂O₂ anddiaminobenzidine tetrahydrochloride [Polysciences, Warrington, Pa.].

Allogeneic MLR

To test for T cell stimulatory function, the APCs were unirradiated orirradiated [Gy 30] and added in graded doses as stimulators for 2×10⁵purified, allogeneic T cells in 96 well flat bottomed plates [Costar].Proliferation was determined on days 4–6 with the addition of 4 μCi/mlof [³H]TdR for 10–16 h to triplicate wells [mean cpm].

Induction of Influenza Virus Specific CTL

Dendritic cells prepared from HLA A2.1+ donors were washed out of mediumcontaining serum and resuspended in RPMI to 0.5–1×10⁷ cells/ml. Live orheat-inactivated influenza virus [PR8, Spafas Inc., Storrs, Conn.] wasadded at a final concentration of 1,000 HAU/ml for 1 hr at 37°, aspreviously described (Bhardwaj et al. 1994; Bender et al. 1995).Following three washes, 3×10⁴ dendritic cells were added to purified Tcells [1×10⁶] in 48 well plates [Costar, Cambridge, Mass.] After 7 days,the T cells were assayed for cytolytic activity on uninfected orinfluenza-infected autologous macrophage targets using a conventional⁵¹Cr release assay (Bhardwaj et al. 1994). Alternatively, targets wereHLA A2.1 matched T2 cells [the TAP deficient line, provided by Dr. P.Cresswell] pulsed for 1 hr with 10 nM influenza virus matrix peptide[GILGFVFTL] (SEQ ID NO:1). Target cells were labeled with Na⁵¹CrO₄ aspreviously described (Bhardwaj et al. 1994).

Results

Cells Cultured in the presence of GM-CSF and IL-4 only were not fullymature. We first confirmed that GM-CSF and IL-4 induces the developmentof large numbers of potent APCs from lymphocyte-depleted PBMC whencultured for 6–7 days in the presence of FCS [2.5×10⁶ per 40 ml ofblood, Table 1 first row].

TABLE 1 Allogeneic MLR ³H-TdR Incorporation, cpm × 10⁻³ AT T:APC ratiosof: Treatment 100:1 300:1 900:1 DC Yield/40 ml blood % Enrichment 10%FCS 153 [17]  33 [3] 7 2.5 × 10⁶  6% 10% FCS + PS  254 [196]  154 [135]79 3.5 × 10⁶ 95% 10% FCS + CM 258 [55]  129 [27] 39 3.8 × 10⁶ 96% 1%Plasma 175 [3]   68 [1] 8 0.5 × 10⁶ 25% 1% Plasma + PS  309 [154]  161[80] 56 2.0 × 10⁶ 75% 1% Plasma + CM 286 [8]  156 [1] 48 1.5 × 10⁶ 74%

Table 1: T Cell Stimulatory Activity of Dendritic Cells Derived fromProgenitors in Blood

ER− cells were cultured for 7 days in RPMI medium containing GM-CSF andIL-4 and either 10% FCS or 1% human plasma. The cells were then culturedfor 4 more days in the absence or presence of conditioned mediumprepared from pansorbin or gamma globulin stimulated blood mononuclearcells [50% final concentration]. T cell stimulatory function wasassessed in the primary allogeneic MLR. Results are shown as ³H-TdRincorporation [cpm×10⁻³] and are averages of triplicates. * Numbers inparentheses represent cpm measured with syngeneic T cells. The yield isexpressed as the total number of dendritic cells obtained from anoriginal donation of 40 ml of blood. The % enrichment is defined as thepercentage of cells in the non-adherent fraction that display typicalfeatures of dendritic cells: stellate shape, high expression of HLA-DR,the dendritic cell restricted marker CD83 [see FIG. 2].

We discovered, however, that dendritic cells primed under theseconditions were not fully mature. Upon immunolabeling of cytospins orcell suspensions [FACS], there was residual expression of the monocytemarkers CD14 and CD32 and weak to no expression of the dendritic cellrestricted markers CD83 (Zhou et al. 1992; Zhou and Tedder, 1995) andp55 (Mosialos et al. 1996).

Furthermore, when FCS primed cells were recultured without cytokines,MLR stimulating activity decreased considerably and most of the cellsattached firmly to plastic [data not shown]. These findings suggested tous that GM-CSF and IL-4 were insufficient triggers to generate matureand stable dendritic cells from non-proliferating blood progenitors.

When 10% human serum or plasma was used in the GM-CSF/IL-4 primingculture in the place of FCS, most of the PBMC remained adherent and fewfree dendritic cells developed by 7 days. To reduce adherence, mediumcontaining 1% plasma or serum was tested instead. Some small floatingaggregates gradually developed over 4 days, and at day 7, some dendriticcells were evident. Although these cells exhibited potent T cellstimulatory capacity in the allo MLR, [Table 1, fourth row], the yieldof dendritic cells was low, only 0.5×10⁶ cells per 40 ml of blood. Aswith FCS, the cells exhibited variable CD14 and CD32 expression andfailed to express CD83 and p55. Thus human plasma was even lessefficient than FCS in priming for mature dendritic cell development.

Requirement for Conditioned Medium

Two approaches were tested to increase dendritic cell yield and maturityin FCS and human plasma. A 1:10,000 dilution of Pansorbin was added tothe cultures after 7 days of growth in GM-CSF and IL-4, and the cellswere recultured for 3–4 days. Pansorbin increased the number of freefloating dendritic cells as well as T cell stimulatory capacity in theallo MLR [Table 1, second and fifth rows). However, there was a dramaticstimulation of syngeneic T cells possibly because of superantigens inthe pansorbin.

In the second approach, we used conditioned medium [CM] derived fromsupernatants of T-depleted mononuclear cells that had been adhered togamma globulin-coated plates or stimulated with pansorbin [see Methods].On day 7 of culture, one half of the medium was replaced with this CM.At day 11, 4 days after the addition of CM, both the yield andenrichment of dendritic cells increased. [Table 1, rows 3 and 6]. Therewas also significant enhancement of allo stimulatory capacity, butwithout the increase in the syngeneic MLR seen with pansorbin. Thus wechose to use CM and 1% human plasma for all subsequent experiments.Plasma is preferable to use over serum since it is readily obtainedduring the Ficoll separation of blood.

In >15 experiments, the overall effect of adding CM to plasma containingmedium was to increase dendritic cell yields 3–10 fold [range of0.3–3×10⁶ per 40 ml of blood] and enhance enrichment from 2–25% to30–90%. The contaminating cells are primarily lymphocytes, B cells andresidual T cells. CM generated cells were phenotypically andfunctionally stable in that they retained their dendritic cell featureswhen cultured in the absence of cytokines [see below]. No substantialdifference was noted whether 33% vs. 50% CM was used or whether the CMwas replenished every 2 days.

Throughout the above cultures, we noted the presence of residualadherent cells, most likely macrophages, in both components of theculture, i.e., during priming for 6–7 d with GM-CSF and IL-4, and duringmaturation for 3–4 d with CM. We therefore modified the protocolfurther. At day 7 of cytokine treatment, we directly transferred thenonadherent cells to fresh plates. We then cultured the cells for 4additional days [day 11] in the absence or presence of CM. As expected,GM-CSF/IL-4 primed cells that were cultured without CM were suboptimalstimulators in the MLR [FIG. 1, closed circles]. In contrast, there wasa substantial increase in stimulatory activity when cells received CM,[open squares]. ER− cells that were cultured in Teflon beakers withoutexogenous cytokines throughout the 11 days were poor APCs [opencircles].

Phenotypic Analysis of Dendritic Cells Grown from Normal PeripheralBlood with GM-CSF, IL-4 and Conditioned Medium

The dendritic cells that were induced by this new culture proceduredisplayed a typical dendritic cell phenotype [FIG. 2] and morphology[FIG. 3]. The cells expressed high levels of MHC class II and accessorymolecules for T cell stimulation [CD54, CD58, CD40, CD80, CD86]. Themore lineage restricted antigens such as CD3, CD19, CD16, CD14 wereabsent. CD1a, CD4, CD11c and CD45RO were also expressed. CD68 ormacrosialin (Ramprasad et al. 1995; Rabinowitz and Gordon, 1991), wasexpressed in a perinuclear pattern. This pattern is distinct from thatseen with macrophages, where staining is seen throughout the cytoplasm.Only rare cells stained with the anti-Lag mAb that identifies Birbeckgranules in epidermal dendritic cells.

Two new markers, CD83 and p55, recently have been shown to beselectively expressed by the small subset of mature dendritic cells incultured human blood (Zhou et al. 1992; Zhou and Tedder, 1995; Mosialoset al. 1995). Both markers were expressed only at low levels by cellsthat are primed with GM-CSF and IL-4, but were unregulated followingculture in CM. CD83 is a member of the Ig superfamily, and p55 is anEBV-inducible actin bundling protein in B cells (Zhou et al. 1992; Zhouand Tedder, 1995; Mosialos et al. 1995).

Stability of Dendritic Cells Generated in GM-CSF/IL-4 and CM

The stability of the dendritic cells generated at various points duringthe two step culture protocol described above was also ascertained. ER−cells were cultured with GM-CSF/IL-4 for 7 days and then analyzed forstimulatory activity in an allo MLR. Although these cells couldstimulate allogeneic T cells [Table 2, first row], the phenotype was nottypical of mature DCs [CD14+, moderate levels of HLA-DR, little to noCD83, and a diffuse staining for CD68; data not shown].

TABLE 2 Allogeneic MLR Treatment ³H-TdR Incorporation Culturesupplements (cpm × 10⁻³) on days: Day of at T:APC ratio of: 0–7 7–1111–14 MLR test 100:1 300:1 900:1 GM + IL-4 — — 7 161 145 81 ″ no CM — 11203 146 25 ″ +CM — 11 154 172 161 ″ +CM no CM 14 144 170 189 ″ +CM +CM14 178 207 165

Table 2: CM is Required for Optimal and Stable ImmunostimulatoryActivity

ER− cells were cultured in GM/CSF and IL-4 for 7 days, washed and thenanalyzed immediately for stipulatory activity in an allo MLR [firstrow], or cultured for 4 more days in the absence or presence of CM[second and third rows]. At day 11, aliquots of dendritic cells weremaintained in CM for three more days [row 4] or washed extensively andrecultured in the absence of CM [row 5]. The same donor of allogeneic Tcells was used throughout these experiments. Results are shown as ³H-TdRincorporation [cpm×10⁻³] and are averages of triplicates.

Cells washed out of cytokines at day 7 and recultured for 4 more days inthe absence of CM had a similar phenotype and a noticeable decline in Tcell stimulatory function when compared with dendritic cells grown inGM-CSF/IL-4 and CM [compare rows 2 and 3, Table 2]. Cells were nextwashed out of CM at day 11 and cultured for three more days [day 14]alongside cells that were left in CM. The dendritic cells were identicalwith respect to their phenotype and stimulatory capacity in theallogeneic MLR [rows 4 and 5, Table 2]. Thus, the exposure to CM rendersan apparent irreversible change that leads to functionally maturedendritic cells.

Attempts to Identify the Active Cytokines in CM

The CM from blood cells that are cultured in human gamma globulin likelycontain many monocyte-derived cytokines such as IL-1, IL-6 and TNFα.However, none of these cytokines could replace the CM, and in fact eachcytokine had no effect on increasing dendritic cell yield or function[FIG. 4 and Table 3]. The cytokines IL-12 and IL-15 were alsoineffective [Table 3].

TABLE 3 Allogeneic MLR Treatment ³H-TdR Incorporation (cpm × 10⁻³)Culture supplements on day 7 at T:APC ratio of: CM Other 100:1 300:1900:1 + goat IgG [4 μg/ml] 147 117 46 + goat IgG [17 μg/ml] 141 94 31 +α TNF-α [1 μ/ml] 144 120 61 + α IL-6 [4 μg/ml] 161 120 31 + α IL-12 ″180 104 43 + α IL-1-α ″ 152 90 33 + α IL-1-β ″ 163 112 37 + cocktail [17μg/ml] 116 59 19 − TNF-α [10 ng/ml] 79 38 11 − IL-1-β ″ 72 47 4 − IL-12[500 pg/ml] 55 21 4 − IL-15 ″ 46 12 4

Table 3: Attempts to Identify the Factors in CM that Promote DendriticCell Differentiation

ER− cells were cultured in GM/CSF and IL-4 for 7 days, and thensupplemented with CM for 4 days in the presence of neutralizingantibodies to a panel of cytokines. Goat IgG was the control. Cocktailrefers to the combination of the various antibodies. Some cultures weresupplemented with recombinant cytokines alone in the absence of CM. Atday 11 of culture the cells were evaluated for their ability tostimulate allogeneic T cells. Results are shown as ³H-TdR incorporation[cpm×10⁻³] and are averages of triplicates.

We also added polyclonal neutralizing antibodies to cytokines to the CM.However, antibodies to TNFα, IL-1, and IL-6 did not block the capacityof CM to increase dendritic cell yields and function (FIG. 4 and Table3].

Source of DC Progenitors in PBMC

The progenitor cells that give rise to dendritic cells producedaccording to an embodiment of the invention [GM-CSF+IL-4 followed by CM]where characterized. To assess the adherence of progenitors to plastic,ER− cells were adhered for 1 hour in 6 well plastic plates [2×10⁶ perwell] after which nonadherent cells were removed and replated in newdishes. Both populations were cultured in GM-CSF and IL-4 for 7 daysfollowed by CM for 4 days. Adherent ER− cells yielded the highest numberand purity of dendritic cells [Table 4]

TABLE 4 DC progenitors are present in both adherent and non-adherentER - fractions. Allogeneic MLR ³H-TdR incorporation (cpm × 10⁻³) atT:APC Cell DC Yield/40 ml % ratio of: population blood Enrichment 100:1300:1 900:1 ER - 0.8 − 1.5 × 10⁶ 79–90 265 243 169 ER -, 1.3 − 2.8 × 10⁶94–96 226 222 147 adherent ER -, 0.8 − 1.7 × 10⁶ 27–36 219 192 73nonadherent

Table 4: Dendritic Cell Progenitors are Present in both Adherent andNonadherent ER− Fractions

ER− cells were adhered to plastic for 1 hr. Both adherent andnonadherent cells were collected and cultured with GM-CSF and IL-4 for 7days, followed by CM. At day 11, the yield and % enrichment of dendriticcells was determined. The T cell stimulatory function of the ER−populations analyzed is also shown. Results are representative of 2experiments and were similar to cells derived from bulk ER− cells interms of immunostimulatory capacity. ER− nonadherent cells alsogenerated some potent APCS, but the yield and purity of dendritic cellswas substantially less, primarily because of a large number ofcontaminating lymphocytes. Thus progenitors appear to reside in bothadherent and nonadherent populations of blood mononuclear cells.

ER− cells were stained with PE-conjugated anti-CD14 and the cells weresorted in a FACStar into CD14 high and CD14 low fractions. Negativesmall cells were excluded by gating. These were then cultured for 7 dayswith GM-CSF and IL-4 followed by 4 days in CM. CD14 high cells were alsoevaluated with or without prior treatment with ionizing irradiation [Gy15 or 30]. The greatest yield, percent enrichment and T cell stimulatoryactivity were found in the unirradiated CD14 high population [Table 5and FIG. 5].

TABLE 5 DC progenitors are enriched in the CD14 hi population Cellpopulation Yield/40 ml blood % Enrichment CD14 hi 1.2 − 2 × 10⁶ 66–97CD14 lo 0.7 − 0.8 × 10⁶   10–39 CDF14 hi, XRT 1 − 1.6 × 10⁶ 72–77 CD14hi + lo 1 − 1.3 × 10⁶ 57–73 [n = 2]

Table 5: Dendritic Cell Progenitors are Enriched in the CD14 HiPopulation

ER− cells were stained with CD14-FITC and sorted into CD14 hi and dimpopulations. Separated fractions, irradiated CD14 hi cells [Gy 15 and30] and stained unsorted or combined cells [CD14 high+low] were culturedwith GM-CSF/IL-4 followed by CM. The yields and % enrichment ofdendritic cells is shown.

Interestingly, irradiated CD14+ high cells generated effective dendriticcells but the yield was 50% lower, and the percent enrichment wasslightly lower, 77% vs. 97% for nonirradiated CD14+ cells. The CD14 lowfraction also produced some typical dendritic cells, but the yield andenrichment were significantly lower than the CD14 high population,probably because there is a significant number of lymphocytescontaminating this fraction.

Collectively, our data suggest that dendritic cell progenitors are foundin PBMC populations that are both plastic adherent and nonadherent, aswell as CD14 dim and CD14 high. Furthermore, some of these progenitorsare radioresistant.

Dendritic Cells Derived from Non-Proliferating Progenitors Induce StrongAnti-Viral Cytolytic T Cell Responses

Potent human CD8+ cytolytic T cell [CTL] responses to live replicatinginfluenza A virus are generated when dendritic cells are the APCs(Bhardwaj et al. 1994). When pulsed with poorly replicating,heat-inactivated, influenza virus, dendritic cells induce equally strongCTL responses (Bender et al. 1995). Other APCs, e.g., macrophages lackthis capacity. Since the heat inactivated virus is incapable ofsubstantial new protein synthesis, only small amounts of viral proteinare required to charge class I molecules on dendritic cells. Dendriticcells prepared as described above from non-proliferating progenitors inblood were tested for their capacity to elicit anti-viral CTL responsesto live and heat-inactivated influenza virus [Table 6].

TABLE 6 Percent specific lysis of targets Infection of Mo Mo dendriticcells [−] [Flu] [−] 2.5 1.9 Live Flu 1.3 60.4 Infection of T2 T2dendritic cells [−] [Matrix peptide] [−] 0 10 Heated Flu 30 77

Table 6: Dendritic Cells Derived from Non-Proliferating ProgenitorsInduce Strong Anti-Viral Cytolytic T Cell Responses

Dendritic cells were uninfected or infected with live replicating orheat inactivated influenza virus [see methods] and cocultured withautologous HLA A2.1+ T cells [T:APC ratio=30:1].

After 7 days, T cells were harvested and tested for cytolytic activityon chromium labeled syngeneic macrophages [mos] which were infected oruninfected with influenza virus [upper panel]. Lytic activity of T cellsresponding to dendritic cells infected with heat inactivated influenzavirus was tested on chromium labeled T2 target cells [lower panel] thathad been pulsed with 10 nM of HLA A2.1 restricted matrix peptide. E/Tratio in both experiments was 30:1.

Both forms of virus could be presented to autologous T cells to induceCTL responses. CD8+ T cells are the likely mediators, since theyefficiently lysed TAP deficient T2 cells pulsed with the HLA A2.1restricted influenza matrix peptide.

Conditions for Generating Mature Dendritic Cells from Progenitors inHuman Plasma

The method of preparing mature dendritic cells according to thisinvention preferably uses two steps. Two steps are preferred becausedendritic cells generated in either FCS or human plasma containingmedium failed to develop and maintain a stable phenotype and functionwhen removed from GM-CSF and IL-4. For example, these “putative”dendritic cells continued to express CD14 and CD32, which are typicallyabsent from mature dendritic cells, expressed little or no CD83 and p55and lost stimulatory function upon removal of GM-CSF/IL-4. Thisphenomenon has not been previously appreciated, since the cells aretypically used without reculturing them in the absence of cytokines(Sallusto and Lanzavecchia, 1995; Sallusto and Lanzavecchia, 1994).Second, exposure to a dendritic cell maturation factor such as CM was anabsolute requisite to induce the formation of large numbers of fullydifferentiated dendritic cells from the GM-CSF and IL-4 primed cultures.The CM-induced cells expressed a typical phenotype, with strongexpression of a] antigen presenting MHC class I and II products andCD1a, b] several adhesions and. co-stimulator molecules including CD40,CD54, CD58, CD80 and CD86, and c] two dendritic cell restrictedmolecules, CD83 and p55. This phenotype remained stable followingremoval of all growth factors. Moreover, the yield and T cellstimulating potency of the CM-treated cells were substantial. Up to3×10⁶ dendritic cells were obtained from 40 ml of blood, which is 3times more than the number of mature dendritic cells that are present inone unit [500 ml] of non treated blood cells. The cells induced strongallo MLRs even at DC:T ratios of 1:900. It is preferred to first primethe blood progenitors with GM-CSF and IL-4 for 6–7 days before addingthe CM, since the latter seems to be less effective if added at day 0[data not shown].

Dendritic Cell Derived from Blood Progenitors Express a Unique Patternof Antigens

We find that CD83 and p55 are useful markers for the development ofmature dendritic cells from blood progenitors. Both antigens areuniformly expressed at high levels in mature cells but are lacking infresh blood monocytes and in progenitors that are primed in GM-CSF/IL-4for 7 days but are not matured in CM. Another useful marker todistinguish dendritic cells from monocytes is CD68 [termed macrosialinin the mouse], a member of the lamp-1 family. This antigen is detectedin a perinuclear area in dendritic cells, whereas much of the cytoplasmstains for CD68 in monocytes, as has been emphasized by Gordon andothers (Rabinowitz and Gordon, 1991; Ramprasad et al. 1995).

Example 2

Materials and Methods

Culture Medium

The following culture media were used: RPMI 1640 [Biological Industries,Kibbutz Beit Haemek Israel]; X-VIVO10, 15, and 20™ [Bio-Whittaker,Walkersville, Md.]; Hybricare and AIM-V™ [GIBCO-BRL, Gaithersburg, Md.].They were supplemented with 50 μg/ml of gentamicin. Sources of serumwere FCS [10%, Biological Industries or Seromed-Biochrom, Berlin,Germany], and human plasma and serum [0.5, 1, 5, and 10%] from eitheradults [PAA Laboratories GmbH, Lin, Austria, blood bank or autologous]or from umbilical cord blood [kindly provided by the Department ofGynecology and Obstetrics]. FSC and, in some experiments only, humansera/plasma was heat-inactivated at 56° C. for 30 minutes.

Cytokines and Anti-Cytokines

Recombinant human GM-CSF was generously provided by Dr. E. Liehi, SandozGes.m.b.H., Vienna, Austria [specific activity 5.9×10⁶ U/mg].Alternatively, human GM-CSF prepared for therapeutical purposes was used[Leukomax™, Sandoz, Basel, Switzerland, specific activity 1.1×10⁷ U/mg].Recombinant human IL-4 was a gift of Dr. M. B. Widmer, Inmmunex Corp.,Seattle, Wash. [specific activity 5×10⁷ U/mg] and Dr. E. Liehl, Sandoz,TNF-α was supplied by Dr. G. R. Adolf [Bender, Vienna, Austria; specificactivity 6×10⁷ U/mg]. IL-12 was provided by the Genetics Institute,Boston, Mass. [specific activity 3.6×10⁸ U/mg]. Human IL-1β [specificactivity 5×10⁸ U/mg] and simian IL-15 [specific activity 1×10⁷ U/mg]were purchased from Genzyme Corp., Cambridge, Mass. human stem cellfactor [specific activity 0.5–1.0×10⁶ U/mg] from R&D Systems [MO]. HumanIL-15 and a neutralizing rabbit anti-human IL-15 antibody were fromPeproTech, London, UK. Human IL-13 was a gift of Dr. A. Minty, Sanofi,Labege, France and was also obtained from Pharmingen, San Diego, Calif.[specific activity 1×10⁶ U/mg].

Initial Processing of Human Blood

Peripheral blood was obtained from the local blood bank as standardbuffy coat preparations and from normal donors. Preservative-freeheparin [200 I.U./ml blood; Novo Nordisk A/S, Bagsvaerd, Denmar] wasused to prevent clotting. PBMC were isolated by centrifugation inLymphoprep [1.077 g/ml; Nycomed Pharma AS, Oslo, Norway]. In order tominimize contamination of PBMC with platelets the Lymphoprepcentrifugation [200×g/room temperature] was interrupted after 20minutes. The top 20–25 ml containing most of the platelets werecarefully removed and centrifugation was resumed [20 minutes/460×g/roomtemperature]. PBMC were then depleted of T and B cells by means of animmunomagnetic technique. Dynaeads [Dynal, Oslo, Norway] M-450 Pan-B/CD19 and M-450 Pan-T/CD2 were washed four times with PBS containing 1%bovine serum albumin or human plasma. PBMC [30–50×10⁶/ml] were incubatedwith the magnetic beads at a ratio of 1:1:1 at 4° C. for 20 min. usingDynal mixer. Lymphocytes were then depleted by means of a Dynal magnet[1–2 minutes]. Magnet-non-adherent cells were harvested and washed. Thisdepletion step was repeated once. In some experiments an indirectimmunomagnetic approach was used. PBMC were labeled with anti-T and Bcell mAb's [hybridoma supernatant of mAb OKT-11/CD2 and ascitic fluid ofmAb MEM-97/CD20; gift of Dr. v. Horejsi, Praha, Czeck Republic], washedthree times and incubated with sheep anti-mouse Ig-coupled magneticbeads [SAM-M450; Dynal] at a ratio of 3 beads to one cell. Magneticseparation was achieved as described above. In addition,lymphocyte-depleted PBMC were obtained from cancer patients [afterinformed consent] in complete remission during hematopoietic recoveryafter high-dose consolidation chemotherapy and subcutaneous dailyadministration of 300 μg G-CSF [Neupogen™, Hoffmann-La-Roche, Basel,Switzerland]. Depletion of PBMC from CD34+ cells was achieved either bymeans of M-450/CD34 magnetic beads [Dynal] or by passing cells throughan immunoaffinity anti-CD34 column [CellPro Inc., Bothell, Wash.] thatis clinically used for transplantation purposes. PBMC from normal donorstreated with G-CSF in the same manner were also used. This study wasapproved by the Ethical Committee of the Medical Faculty, University ofInnsbruck.

Culture Technique

Lymphocyte-depleted PBMC were planted in 6-well tissue culture plates ata density of 2×10⁶ cells/well in 3 ml of complete culture medium. GM-CSFand IL-4 were added at final concentrations of 800 and 1000 U/ml,respectively. Cultures were fed every other day [days 2, 4 and 6] byremoving 1 ml of the medium and adding back 1.5 ml fresh medium withcytokines [1600 U/ml GM-CSF and 1000 U/ml Il-4, resulting in finalconcentrations of 800 and 500 U/ml, respectively]. On day 7,non-adherent cells were either harvested and analyzed or transferred tonew 6 well plates and cultured further in the presence or absence ofmaturation stimuli [see below]. GM-CSF and IL-4 were present during theculture period from day 7 to day 10–11.

Stimuli for Dendritic Cell Maturation

SACS [fixed Staphylococcus aureus Cowan 1 strain, 2.01 mg/ml Ig-bindingcapacity, Pansorbin cells, Cat No. 507861] was purchased from CalBiochemCo., La Jolla, Calif. It was added to dendritic cell cultures at a finaldilution of 1:10000. Conditioned medium was produced by Ig-adherentPBMC. To this end, bacteriologic plates [100 mm. Cat. No. 1029, Falcon,Oxnard, Calif.] were incubated with a solution of human Ig [10 mg/ml,therapeutically used “Immunoglobulin i.v. Biochemie”, BiochemieGes.m.b.H., Vienna, Austria, subsidiary of Sandoz] in PBS for 1 minuteat room temperature. After three rinses with PBS 5×10⁷ PBMC were putinto one Ig-coated bacteriologic plate for 1 hour at 37° C. in 8 ml ofcomplete culture medium. Non-adherent cells were rinsed away andadherent cells were cultured in fresh complete medium at 37° C. for 24h.

Complete media consisted of the different media and serum supplementsmentioned above. Conditioned media were sterile filtered and stored at+4° C. for one week at the longest or frozen at −20° C. In some cases,conditioned medium was prepared from cells stimulated with SCS insteadof human Ig. Conditioned media were routinely used at 25% vol/vol.Increasing this concentration up to 50% was not appreciably better.

Flow Cytometry and Immunohistochemistry

Primary antibodies used for flow cytometric and immunohistochemicalanalyses are listed in Table 7.

TABLE 7 Antibodies used for immunohistochemistry Specificity Clone/NameIg class Source Reference HLA-DR/DQ 9.3F10/HB 180 mouse IgG2a ATCC^(a)HLA-DR L243/HB55 mouse IgG2a ATCC HLA-DR-FITC L243 mouse IgG2a BDIS^(b)CD1a OKT-6/CLR8020 mouse IgG1 ATCC CD1a-FITC OKT-6 mouse IgG1 Ortho^(c)CD15s CSLEX1 mouse IgM BDIS CD40 G-28.5 mouse IgG1 E. A. Clark⁴ CD44v9VFF16 mouse IgG P. Herrlich^(c) CD45RA 4G10 mouse IgG2a R. M.Steinman^(f) CD45RO UCHL10 mouse IgG2a DAKO^(g) CD68 EBM11 mouse IgG1DAKO CD71 RPN.511 mouse IgG1 Amersham^(h) CD80 L307.4 mouse IgG1 BDISCD83 HB-15a mouse IgG2b T. F. Tedder^(i) (Zhou et al. 1992) CD86 IT2.2mouse IgG2b Pharmingen^(j) CD115/c-fms Ab-1 rat IgG1 Oncogene Ashmun etal., 1989 CSF-1-receptor (clone 2-4A5-4) rat IgG2b Science^(k) Ab-2(clone 3-4A4-E4) Birbeck granules Lag mouse IgG1 S. Imamura^(l)(Kashihara et al. 1986) Proliferation- Ki-67 mouse IgG1 DAKO associated

TABLE 7 Antibodies used for immunohistochemistry a American Type CultureCollection, Rockville, MD; b Becton-Dickinson Immunocytometry Systems,Mountain View, CA; c Ortho, Raritan, MJ; d Seattle, WA; eKernforschungszentrum, Karlsruhe, Germany; f The Rockefeller University,New York, NY; g Roskilde, Denmark; h Amersham, UL; i Duke University,Durham, NC; j San Diego, CA; k Cambridge, MA; l Kyoto University, Kyoto,Japan

Secondary antibody was biotinylated anti-mouse or anti-rat lg's followedby FITC- or PE-conjugated streptavidin [Amersham International,Amersham, UK]. Dead cells were gated out on the basis of theirflorescence with propidium iodide or by their light scatter properties.Analyses were done on a FACScan instrument [Becton-Dickinson, MountainView, Calif.]. Cytospins were prepared with Cytospin 2 centrifuge[Shandon, Inc., Pittsburgh, Pa.]. Slides were acetone-fixed for 5 min.at room temperature and then incubated with primary mAbs for 15 minutesfollowed by biotinylated sheep anti-mouse Ig [1:200; Amersham] and TexasRed-conjugated streptaviden [1:50, Amersham]. For double labelingpurposes this staining sequence was extended with a blocking step [100μg/ml mouse gamma globulin] and FITC-conjugated anti-HLA-DR[Becton-Dickinson]. Slides were mounted in Vectacshield™ [VectorLaboratories, Burlingame, Calif.]

Allogenic MLR and Processing/Presentation of Soluble Antigen

To test for T cell stimulatory function, the APCs were irradiated with30 Gy from a Cs source and added in graded doses as simulators for 2×10⁵purified [nylon wool-non adherent; anti MHC class II-panned], allogeneicT cells in 96 well flat bottomed plates [Falcon]. For some experiments Tcells were purified from umbilical cord blood in order to obtain naive Tcells. Proliferation was determined on days 4–6 with the addition of 4μCi-148 KBq/ml of [³H]TdR [specific activity 247.9 GBq/mmol=6.7 Ci/mmol;New England Nuclear, Boston, Mass.] for 12–16 h to triplicate wells[mean cpm]. Processing and presentation of tetanus toxoid protein[Connaught, Willowdale, Ontario, Canada] was measured with aHLA-DP4-restricted tetanus-peptide-specific T cell clone [AS11.15] thatwas a gift of Dr. A. Lanzavecchia, Basel Switzerland (Lanzavecchia,1985). Graded doses of irradiated dendritic cells were co-cultured with1.5×10⁴ clone cells in the presence or absence of tetanus toxoid [5 and1 μg/ml]. [³H] TdR was added from d2 to d3.

To generate cytotoxic T cells, the approach described by (De Bruijn etal. 1992) was used with modifications. Irradiated [12.5 Gy] dendriticcells as simulators were co-cultured with autologous PBMC or with CD8+ Tlymphocytes [1:25] as responders in the presence of 100–200 μM peptideMP 58–66 of influenza matrix protein [MedProbe, Oslo, Norway] in 2 mlRPMI/10% human serum in wells of a 24-cell tissue culture plate. Afterone week the cultures were fed with 40% Lymphocult [Biotest, Dreieich,Germany]. The lytolytic potential of outgrowing T cells was assessed twoto four weeks after the start of the cultures in a standardChromium-release cytotoxicity assay using as target PHA-stimulatedautologous T cell blasts, that had been incubated with differentconcentrations of the peptide. Results are expressed as % specificlysis. CD8+ T lymphocytes were prepared by indirect immunomagneticdepletion of CD4+ T cells using mAb VIT-4/anti-CD4 [gift of Dr. W.Knapp, Vienna, Austria] followed by sheep anti-mouse lgG magnetic beads[Dynal].

Additional Reagents

Latex beads [0.5% vol/vol; 2 μm diameter] for phagocytosis experimentswere purchased from Seradyn, Indianapolis, Ind. They were added to thecell cultures at a final dilution of 1:20. Staphylococcal enterotoxin A[superantigen SEA] was from Sigma Chemical Corp. St. Louis, Mo. It wasused at a final concentration of 25 μg/ml. LPS [Sigma] was added to someexperiments at a final concentration of 100 ng/ml.

Results

Identification of Culture Conditions Allowing full Maturation ofDendritic Cells

Dendritic cells grown in GM-CSF and IL-4 are not fully mature.Lymphocytes were depleted from PBMC by means of immunomagnetic beads.Use of the immunomagnetic beads allowed for the omission of a sensingstep two hours after plating. Dendritic cells developed from theselymphocyte-depleted PBMC in the same way as from whole PBMC populations.

Dendritic cells, that had been washed out of GM-CSF and IL-4 and werereplated in culture medium without cytokines for an additional threedays [days 7–10], lost their characteristic morphology and re-adhered.They assumed the typical shapes of macrophages [FIG. 6A]. In addition,dendritic cells on d7 of culture expressed the macrophage marker CD 115[c-fms/CSF-1 receptor] although their CD14 expression was very low orabsent. In contrast, they did not express CD83, a marker for maturedendritic cells (Zhou and Tedder, 1995) [FIG. 7]. The staining patternof a well defined population of mature dendritic cells, namely cutaneousdendritic cells obtained by emigration from skin organ cultures (Pope etal. 1995) was inverse: CD115/CD83+ [not shown]. From this we concludedthat dendritic cells grown in GM-CSF and IL-4 were not fully andirreversibly mature although they did express some maturation markerssuch as high levels of MHC class II, furthermore CD40, 54, 58, 80 and astrong T cell stimulatory capacity (Romani et al. 1994; Sallusto andLanzavecchia, 1994).

Determination of Stimuli for full Maturation of Dendritic Cells

We tested whether full maturation could be brought about by addingcytokines to the cultures. Supplementation of GM-CSF and IL-4 containingculture medium with IL-1β [50 U/ml] and/or TNF-α [50 U/ml] either fromday 0 through to d7 or as a 24-hour pulse from day 0 to day 1 or day 6to day 7 increased immunostimulatory capacity as expected but did notlead to a stable phenotype of mature dendritic cells as defined above.Addition of these cytokines after the initial 7-day culture and transferof the cells to fresh culture wells [i.e., from day 7–10] gave the sameresult [FIG. 8]. Similar findings were made for the cytokines IL-12 [1ng/ml], stem cell factor/c-kit ligand [yyU/ml] and IL-15 [20 ng/ml].

SACS [fixed Staphyloccus aureus], a potent natural stimulator ofcytokine secretion was also tested. When a 1:10,000 dilution of SACS wasadded to the cultures from day 7 to day 10 or 11 of culture, the numbersof free floating dendritic cells increased markedly. Moreover, thesecells displayed a more pronounced morphology with highly motilecytoplasmic processes [“veils”]. The cells also did not re-adhere whenreplated without cytokines. T cell stimulatory capacity in theallogeneic MLR was superior to dendritic cells that had been culturedwithout SACS [FIG. 8]. However, there was a dramatic stimulation ofsyngeneic T cells possibly because of superantigens in the SACSpreparation. Addition of superantigen to the cultures between days 7 to10 did not bring about maturation of dendritic cells. However,supernatants of SACS-stimulated adherent PBMC [i.e., conditioned media]had the same effect as SACS itself.

Conditioned medium [CM] was produced in a way that could potentially beof clinical use and avoid the involvement of bacterial products such asSACS. Therefore, we derived supernatants from whole or T-depletedmononuclear cells that had been stimulated by adherence to Ig-coatedplates. On day 6 to 8 [mostly 7] of culture dendritic cells weretransferred to fresh wells and 25% vol/vol of CM was added. Three tofour days later the cells were analyzed. There were almost no adherentcells left on the bottom of the wells. By phase contrast microscopyrevealed that the floating cells had many motile veils, much likeSACS-treated cells [FIG. 9]. In the hemocytometer the cells appeared“hairy” like cultured epidermal Langerhans cells or cutaneous emigrantdendritic cells. This is in contrast to either 7 day or 10 day cultureswithout CM, where the cells are large with irregular outlines, but thenumber of long processes and veils is low. Cultured without cytokinesthe CM-exposed cells remained stably non-adherent over an observationperiod of 3 days [FIGS. 6B–D]. They now expressed CD83 and were highlystimulatory for resting T cells [see below]. Therefore, dendritic cellsgenerated in the presence of monocyte CM qualified as fully maturedendritic cells. In >15 experiments the yields of mature,CD83-expressing dendritic cells per 40 ml of blood ranged between 1 and4×10⁶ in FCS-containing media. Maturation occurred likewise when IL-4was replaced with IL-13 [20 ng/ml].

To ascertain whether the activity of CM is due to IL-15, a neutralizingantibody was added to CM during the maturation phase of culture.Maturation was not prevented or impaired. We also measured whether CMcontained bioactive IL-15. IL-2-responsive CTLL-2 cells alsoproliferated vigorously in response to both human and simian IL-15 inconcentrations as low as 5 pg/ml. No response was seen when CM weretested in this assay [n=2] indicating that IL-15 is present only inminute quantities if at all [Table 8].

TABLE 8 Test for IL-15 in the monocyte conditioned medium anti-IL-15Additions to the CTLL no anti-IL-5 anti-IL-15 1:100 + bioassay for IL-15antibody 1:100 1:200 excess IL-15 no cytokines 0.2 IL-15 [500 pg/ml]152.8 148.8 161.1 149.2 IL-15 [50 pg/ml] 151.1 145.3 147.0 144.2 IL-15[5 pg/ml] 45.8 0.9 0.8 156.9 monocyte conditioned 0.6 2.4 1.1 155.0medium [25% vol/vol]

Table 8: IL-15-dependent proliferation of CTLL-2 cells was determined bymeasuring ³H-TdR incorporation during the final 6 hours of a 36 hourculture [6×10³ CTLL cells per well]. Values are means of triplicatewells and are given in cpm×10⁻³. Excess IL-15 was added at a finalconcentration of 1 ng/ml.

Development of FCS-free Media

Having established the conditions for the full maturation of dendriticcells it was desirable to develop a system without fetal calf serum.Simply replacing FCS with human pool sera resulted in little or nodevelopment of dendritic cells. We observed increased adherence of cellsto the culture surfaces. Serum, plasma and serum from umbilical cordblood were tested over a concentration range from 0.5 to 10%. We alsocompared autologous sera/plasma versus pooled sera/plasma from the bloodbank or commercially available reagents. The effects ofheat-inactivation were tested. These variables were combined withdifferent culture media such a RPM1640, X-VIVO, AIM-V, Hybricare, andIscove's . These reagents were used for the culture of both progenitorcells and CM-producing adherent PBMC. The primary read-out system wasmorphology under the inverted phase contrast microscope and the yield ofmature, stable dendritic cells. Cultures in RPMI supplemented with 10%FCS were always run in parallel as a positive control. RPMI1640containing 1% autologous, not heat-inactivated human plasma was observedto be optional. In 12 standardized experiments the yield ranged between0,8 to 3,3×10⁶ CD83+ dendritic cells from 40 ml of blood. Enrichment ofCD83+ cells was 30 to 80%.

Properties of Mature Dendritic Cells

Phenotype. FACS analyses were performed before and three days afterexposure to CM. Alternatively, dendritic cells were cultured from day 7to day 10 in the presence or absence of CM and then compared to eachother. FIG. 7 shows expression of the key markers: CD83 is induced, CD86is enhanced, and CD115 is lost. This pattern applies also to dendriticcells cultured in clinically approved media and derived from PBMC thathave been depleted from lymphocytes by immunomagnetic beads or sheeperythrocyte resetting. CD14 is low or absent and CD1 a shows somereduction in expression levels upon maturation. Further CM-inducedphenotypical changes include the down-regulation of CD32 and CD45RA andthe upregulation of CD45RO. Substantial levels of CD4 are expressed onboth immature and mature dendritic cells. Staining of cytocentrifugesmears [not shown] also reflected the maturation event that takes placeunder the influence of CM. Dendritic cells on day 7 of culture arestained throughout the whole cytoplasm in a pattern typical formonocytes or macrophages. After three days of culture in the presence ofCM the immunostained cellular structures become concentrated in a dullspot near the nucleus. This pattern has been described for maturedendritic cells. MAb's Lag and Ki-67, specific for Birbeck granules ofepidermal dendritic cells [i.e., Langerhans cells] and aproliferation-associated antigen, respectively, stained only raredendritic cells.

Function. One of the mechanisms by which dendritic cells take upantigens is phagocytosis. This function is down-regulated upon cultureof dendritic cells (Reis e Sousa et al. 1993). We tested the phagocyticcapacity of dendritic cells before and after exposure to CM. Immaturedendritic cells readily took up 2 μm latex beads; mature dendritic cellsafter three days of culture in CM phagocytosed only very few particles[FIG. 10].

Maturation in conditioned medium enhanced the stimulatory capacity forresting T cells markedly (FIGS. 11A, 11B). Cord T cells, i.e., naive Tcells were also efficiently stimulated by mature dendritic cells [FIG.11C]. When we tested for the capacity to process and present a solublenative protein antigen [tetanus toxoid] using a tetanus peptide-specificT cell clone a reciprocal pattern emerged. Dendritic cells that had beencultured in the presence of CM were less efficient than those culturedin the absence of CM [FIG. 11D] indicating maturation (Sallusto andLanzavecchia, 1994; Romani et al. 1989a). Mature dendritic cells, whenpulsed with a dominant influence matrix peptide that is presented onHLA-A2.1 were able to elicit peptide-specific, MHC class I-restricted,lytic T cells lines from autologous PBMc or populations of CD+ T cells[FIG. 11E, F].

Modifications of the Method with Regard to Clinical Applicability

With regard to the clinical application of cultured dendritic cells wetested whether the method would work in the complete absence ofxenoproteins and with culture media that are approved for clinical use.Comparative data showed that depletion of lymphocytes [T and B] withimmunomagnetic beads was equivalent to the commonly used method ofresetting with neuraminidase-treated sheep erythrocytes discussed above.Similar numbers of stimulatory dendritic cells were obtained. Anti-B andT cell mouse mAb's and immunomagnetic sheep anti-mouse lg beads, bothapproved for clinical use are available from Baxter Healthcare Corp.[Glendale, Calif.]. Culture media for clinical use are optimized for thegrowth of cell types other than dendritic cells [e.g. tumor-infiltratinglymphocytes]. Yet, in more than 15 experiments mature dendritic cellscould consistently be procured. When X-VIVO 20 medium supplemented with1% autologous human plasma was used for both culturing the cells andproducing CM, substantial numbers of stimulatory dendritic cells couldbe obtained. Yields and percentages of enrichment were, however, onlyabout half of what could be achieved with RPMI 1640 medium [n=5]. Moreadherent cells were observed in these cultures. Pilot experiments withAIM-V medium gave similar results. X-VIVO 10 and X-VIVO 15 media yieldedunsatisfactory results.

With regard to the possibility that only small volumes of blood may beavailable in certain clinical settings we determined that pretreatmentof donors with G-CSF increased dendritic cell yields. Blood from G-CSFtreated cancer patients and normal individuals gave up to 6-fold yieldsof mature [i.e., CD83+] dendritic cells. This increase was not only dueto an increased frequency of CD34+ precursor cells in the blood becausesimilar numbers of immunostimulatory mature dendritic cells could begrown both from CD34-containing and CD34-depleted PBMC startingpopulations in response to GM-CSF, IL-4 and CM [FIG. 12].

We also tested whether cryopreservation of dendritic cells precursorsand their progeny is possible. Indeed, using standard freezingprocedures dendritic cells could be generated from thawed populations ofPBMC. Although not investigated systematically, it is preferred tofreeze the starting population of PBMC or immature dendritic cells onday 7 of culture [i.e.], the day of transfer into CM-containing mediumrather than mature cells on day 10/11 of culture.

The criteria used initially to define fully mature dendritic cellsincluded: morphology, lack of adherence to plastic, and select surfacemarkers. Dendritic cells grown for six days in GM-CSF and IL-4, i.e.,immature dendritic cells did not express CD83 but did express CD115[CSF-1 receptor/M-CSF receptor]. When they were replated withoutcytokines [and without CM] they adhered to the bottom of the culturewells. After a three day culture in the presence of CM they becameCD83+/CD115− and did not become adherent upon withdrawal of cytokines.They also maintained their pronounced cytoplasmic, motile “veils”. Theseresults are consistent with the production of fully and stably maturedendritic cells. Only SACS, conditioned media from SACS-stimulatedadherent PBMC, and conditioned media from monocytes stimulated byadherence on Ig were able to make dendritic cells reach full maturation.When we tried to replace CM with cytokines we observed no effects orsome increase in T cell stimulatory capacity [with IL-1β, and/or TNF-αor IL-15]. Full maturation was not achieved, however. In as shown inExample 1, neutralizing antibodies to IL-1α and β, IL-6, TNF-α, IL-12,alone or in combination, do not block the activity of the monocyte CM.CM may contain either a unique cocktail of cytokines that is difficultor impossible to reproduce experimentally or new cytokines.

Example 3

Clinical Use of Dendritic Cells Primed with Influenza or HIV Antigens

In this Example, dendritic cells prepared as described in Example 1 areprimed with peptides that are presented on MHC class I molecules toprime CD8+ T cells. We have used the immunodominant influenza matrixpeptide, GILGFVFTL, that is recognized by CTLs from HLA-2.1 individuals,and we will in addition use HIV-1 peptides [pol ILKEPVHC-V; gagSLYNTVATL] that are recognized by CTLs from HIV-1 infected individuals.The efficacy of the peptide pulse can be monitored by applying analiquot of the DCs to autologous T cells in culture and measuring thekiller cell response.

Most humans are primed to influenza antigens, so that the recall CTLresponse to this virus provides an optimal and internal control todetermine if autologous DCs, pulsed ex vivo with viral antigens, canboost the CTL response in vivo.

In this protocol, HIV-1 infected individuals are treated with CD4+ Tcell counts of 300–400/ul. These individuals have low numbers of HIV-1specific, killer cell precursors. Asymptomatic, HIV-1 infected, adultvolunteers of both sexes will be recruited. The donors must be HLA-A2.1positive, as will be determined by initial typing with a monoclonal toA2.1. Donors who have relatively low and high recall responses toinfluenza will be included. The therapeutic goal is to increase thenumbers of these killers in order to reduce the cell-cell spread ofHIV-1. The killer cell response to influenza is an internal control,since it should be intact in HIV-1 infected patients.

Pairs of HIV-1 peptides are used so that the response to dendritic cellsadministered by two routes, intradermal [i.d.] and subcutaneous [s.c.]can be compared in the same patient. The influenza peptide will serve asthe common denominator for each route. The peptides are made accordingto Good Laboratory Practices [GLP] at Sloan Kettering Institute, wherepeptides have been synthesized previously for administration in humans.

The Clinical Proctocal is as follows:

-   a. Take a 50–60 ml blood sample in heparin by venipuncture.-   b. Isolate the mononuclear cells on Ficoll Hypaque, and save the    autologous plasma.-   c. Separate the mononuclear cells into T cell enriched and T cell    depleted fractions by sheep erythrocyte resetting, or by depleting    lymphocytes with GMP-grade DYNAL beads coated with monoclonal    antibodies to B and T cells. The T cells are frozen for tissue    culture tests for antigen presentation to cytolytic T lymphocytes    [CTLs, below].-   d. Culture a fraction of the mononuclear cells on culture plates    that have been coated with human Ig [Sandoz] in an RPMI-1640 culture    medium supplemented with antibiotics [penicillin and streptomycin].    This stimulates the cells to form a conditioned medium that fosters    DC maturation.-   e. Culture the bulk of the T-depleted mononuclear cells for 6 days    in RPMI-1640 supplemented with antibiotics, 1% autologous plasma,    1000 U/ml rHuGM-CSF, and 1000 U/ml rHuIL-4. GMP grade cytokines are    provided by Schering-Plough.-   f. After 6 days, transfer the cells to fresh medium supplemented    with 30–50% v/v autologous monocyte conditioned medium plus 1 uM of    influenza matrix peptide, GILGFVFTL (SEQ ID NO:1). One aliquot of    the DCs is pulsed with the HIV-1 gag peptide and the other with the    polymerase peptide. At day 11, when the DCs have acquired their    typical stellate shapes, the DCs are harvested, washed, counted, and    readied for injection by s.c. and i.d. routes. Prior to the    injection, the patients are typed to be sure that they have killer    cell precursors specific for the above peptides.-   g. The DCs are injected in the forearm. On one side the cells are    given in a total of 0.5ml in 3 intradermal sites, while on the other    side, the cells are given in 0.5 ml in 3 subcutaneous sites. The    purpose of the two routes is to compare the efficacy of boosting to    the influenza matrix peptide.-   h. 1 week and 1 month after injection, 25 ml of blood is taken to    evaluate the induction of immune responses. T cell responsiveness to    the peptide is measured by antigen-induced proliferation and release    of IFN-gamma in culture. The results are compared to preinjection    values which are obtained 0 and 1 month prior to injection. The    first injection utilizes DCs that are not pulsed with peptide. T    cell responsiveness to peptide will be measured by enumerating CTL    precursor frequencies [CTLp] for the peptide-coated, HLA-A2.1 “T2”    cell line.-   i. If priming is not detected at 1 month, a second dose of antigen    pulsed DCs is be administered. If priming is detected at 1 month,    the immune status is reexamined at 3 months and 6 months to    determine the longevity of immune memory.

While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that the basic constructions can be altered toprovide other embodiments which utilize the methods and compositions ofthe invention. Therefore, it will be appreciated that the scope of thisinvention is defined by the claims appended hereto rather than by thespecific embodiments which have presented hereinbefore by way ofexample.

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All of the foregoing are incorporated herein by reference.

1. An in vitro method for producing stable mature dendritic cells frompluripotential cells, comprising: a) contacting said pluripotentialcells having the potential of expressing either macrophage or dendriticcell characteristics with a combination of GM-CSF and IL-4 or IL-13 fora time sufficient to produce immature dendritic cells, and b) adding acomposition selected from the group consisting of peripheral bloodmononuclear cell conditioned medium, monocyte conditioned medium andfixed Staphylococcus aureus Cowan 1 strain (SACS) to the immaturedendritic cells produced in step (a) and culturing the cells for a timesufficient for the immature dendritic cells to produce stable maturedendritic cells that express a characteristic of mature dendritic cells,wherein the characteristic is selected from the group consisting ofincreased CD83 expression, increased CD86 expression, decreased CD115expression, and decreased CD32 expression relative to the immaturedendritic cells.
 2. The method of claim 1, wherein the pluripotentialcells are CD14 positive mononuclear pluripotential cells.
 3. The methodof claim 1, wherein the pluripotential cells are peripheral bloodmononuclear cells.
 4. The method of claim 1, wherein the pluripotentialcells are monocytes.
 5. The method of claim 1, wherein the compositionfurther comprises GM-CSF.
 6. The method of claim 5, wherein thecomposition further comprises IL
 4. 7. The method of claim 1, whereinthe GM-CSF is present at a concentration of between about 200 U/ml toabout 2000 U/ml.
 8. The method of claim 1, wherein the dendritic cellsexpress high levels of MHC class molecules.
 9. The method of claim 1,wherein the dendritic cells have the capacity to stimulate resting Tcells.